Process and reagent for the determination of substrates or enzyme activities

ABSTRACT

A process for the determination of substrate or enzyme activities by the use of a redox reaction as a measurement reaction is carried out in the presence of one or more additionally added tetrazolium salts to remove disturbing substances. The tetrazolium salts have the formula ##STR1## in which R 1  is a hydrogen atom, a carboxyl group or an alkyl, phenyl, nitrophenyl, dinitrophenyl, carboxyl-substituted phenyl or trialkylammoniumphenyl radical, R 2  is a phenyl, nitrophenyl, biphenylyl or naphthyl radical, R 3  is a phenyl, carboxyl-substituted phenyl, carboxyl-substituted hydroxyphenyl or dimethylthiazolyl radical, and A.sup.⊖ is a monovalent anion. The formazanes formed by reaction with reducing substances do not absorb light at all, or absorb light only to a negligible extent, at the measurement wavelength of the redox reaction. 
     New compounds included within the structural formula are those in which R 1  is carboxy, phenyl, 2-carboxyphenyl, 4-carboxyphenyl, 2,4-dinitrophenyl or 4-trimethylammoniumphenyl, R 2  is phenyl, and R 3  is phenyl or 2-carboxyphenyl; with the proviso that, when R 3  is unsubstituted phenyl or 2-carboxyphenyl, R 1  may not be unsubstituted phenyl.

This is a divisional of application Ser. No. 035,617 filed on Apr. 3,1987, now U.S. Pat. No. 5,013,647.

The present invention is concerned with a process and reagent for thequantitative determination of substrates or of enzyme activities withthe use of a redox reaction as measurement reaction.

In clinical and pharmaceutical chemistry, in biochemistry and infoodstuff chemistry, a number of indicator methods are used for thedetermination of substrates or of enzyme activities. However, ofespecial important is, for example, the change of the extinction of aredox color indicator system or changes in the electrical potential orcurrent which can be measured by a suitable electrode. Such redoxreactions are especially widely used in clinical chemistry.

If, in the test system used for the photometric determination of aparticular substrate or enzyme which depends upon the use of a redoxreaction as measurement reaction, besides the interesting redoxcomponents, other reducing substances are also present, thendisturbances of the measurement reaction are to be expected.

As is known, especially in the case of the use of biological samplematerials, such as urine, plasma or serum, which can contain reducingsubstances not only of endogenic but also of exogenic origin insignificant amounts, such disturbances can be expected. In the case ofendogenic disturbing substances, bilirubin is to be especiallymentioned. Disturbing reducing substances of exogenic origin can be, forexample, ascorbate and various medicaments or metabolites thereof.Medicaments which occur frequently in biological samples and whichdisturb because of their reducing properties include, for example,α-methyldopa and dobesilate calcium. As an example of a disturbingmetabolite, there may be mentioned homogentisic acid.

Disturbances due to reducing substances have a particularly markedeffect when the concentration or activity of the substrate or enzyme tobe determined in the sample is relatively low and, for the achievementof a sufficient measurement precision, a relatively high sample/reagentvolume ratio is necessary. This is, for example, the case in thedetermination of uric acid, creatinine or oxalate in serum or plasma. Inthese cases, a substrate to be determined is reacted with an enzyme withthe formation of hydrogen peroxide, the resultant hydrogen peroxidebeing utilized in the presence of peroxidase in a stoichiometric coloredmaterial formation reaction, for example the oxidative coupling of twocoupling components.

Hitherto, it was not possible to remove disturbances due to reducingsubstances quickly and also effectively and without additionalimpairment of the redox reaction necessary for the measurement. For thispurpose, in chemistry strongly oxidizing substances are usual. However,for enzymatic methods of determination in clinical and pharmaceuticalchemistry, in biochemistry and in foodstuff chemistry, these cannot beused since they also attack and destroy substrates and enzymes.Furthermore, they frequently lead to side reactions which increase theextinction of the blank.

Many metal salts and complexes thereof also react with reducingsubstances with the formation of the corresponding di- and trivalentions. These frequently inhibit the enzymes which are used for theindicator reaction. Furthermore, the resultant reduced metal ions candisturb in the same way as the disturbing substances present in thesample material. An additional disadvantage of metal ions is that theoxidation thereof is optimum under strongly acidic conditions. Underthese conditions, enzymes are usually destroyed.

Apart from these conventional processes, especially for ascorbic acidthere is the rapid oxidative decomposition of ascorbate with atmosphericoxygen. However, this is only possible under strongly alkalineconditions under which enzymes are denatured or inactivated.

As an alternative, the use of ascorbate oxidase as a catalyst for thisreaction has been suggested. See U.S. Pat. No. 4,168,205.

Because of the specificity of the enzyme ascorbate oxidase, apart fromascorbate this process cannot be used for other reducing disturbingsubstances. The pH optimum of the enzyme is 5 to 6 and thus is farremoved from the pH optima of many enzymes which are important forclinical tests. Thus, for example, the determination of creatinine or ofuric acid takes place at pH 8. At this pH value, the activity ofascorbate oxidase is not sufficient for a rapid removal of disturbances.Furthermore, this enzyme is inhibited by some conventional testcomponents, such as azides, or chelating agents, such asethylenediamine-tetraacetic acid.

Therefore, there was a need for a universally usable process and reagentfor the determination of substrates or of enzyme activities whichremoves disturbances due to reducing substances in redox reactionswithout, however, disadvantageously influencing the measurementreaction. In addition, the reagent should be easily to use and also beeconomical. It was an object of the present invention to satisfy thisneed.

This object was achieved by the process according to the presentinvention for the determination of substrates or enzyme activities inwhich, when a redox reaction is used as measurement reaction, working iscarried out in the presence of one or more additional added tetrazoliumsalts.

The advantage of this process consists in the universal, irreversible,simple and rapid removal of reducing substances which disturb under theconditions prevailing in the substrate or enzyme test in question. Inthis way, a disturbance-free measurement is made possible. Tetrazoliumsalts and the reduction thereof to formazanes have already been knownsince the end of the last century. They are preponderantly used for thedetection of reducing substances in chemistry, biochemistry andhistochemistry (cf.: Bergmeyer, Methods of Enzymatic Analysis, Volume I,pp. 199 et seq./1984; and F. P. Altmann, Progress in Histochemistry, 9,p. 1 et seq./1976). Hitherto, however, it has never been considered tocombine a redox reaction acting as a color indicator with a tetrazoliumsalt reduction with the object of utilizing this reduction of atetrazolium salt to a formazane not as a detection reaction but as areaction for removing disturbances. The previous interest has been tofind tetrazolium salts which, upon reduction, form formazanes which areas colored as possible and, therefore, can be used as color indicatorsin redox reactions.

In order not to disturb the color indicator system in a redox reactionwhich can be monitored photometrically and is used as a measurementreaction, the tetrazolium salts used for the removal of disturbingsubstances must form formazanes which absorb no light at all or only toa negligible extent at the measurement wavelength of the actual colorindicator system. For the color indicator systems which are, inparticular, usual in clinical chemistry with maximum wavelengthabsorptions of from 500 to 600 nm, according to the present invention,there can, in principle, be used any tetrazolium salt which does notabsorb in this wavelength range or only absorbs to a negligible extent.

Within the scope of the present invention, tetrazolium salts which haveproved to be especially useful are those of the general formula:##STR2## wherein R¹ is a hydrogen atom, a carboxyl group or an alkyl,phenyl, nitrophenyl, dinitrophenyl, carboxyl-substituted phenyl ortrialkylammonium-phenyl radical, R² is a phenyl, nitrophenyl, biphenylylor naphthyl radical, R³ is a phenyl, carboxyl-substituted phenyl,carboxyl-substituted hydroxyphenyl or dimethylthiazolyl radical andA.sup.⊖ is a conventional counterion.

An alkyl radical in the definition of R¹ contains 1 to 10 and preferablyup to 7 carbon atoms, the methyl, ethyl and n-butyl radicals beingespecially preferred.

The nitrophenyl radical in the definition of R¹ and R² is especiallypreferably a p-nitrophenyl radical.

The dinitrophenyl radical mentioned in the definition of R¹ ispreferably a 2,4-dinitrophenyl radical.

Carboxyl-substituted phenyl radicals in the definitions of R¹ and R³ arepreferred when the phenyl radical is substituted in the 4-position by acarboxyl group. Furthermore, in the definition of R³, those radicals areespecially preferred when the phenyl radical carries the carboxylsubstituent in the 2-position.

A trialkylammoniumphenyl radical in the definition of R¹ is especiallyadvantageous when the phenyl radical carries the trialkylammoniumsubstituent in the 4-position. The alkyl radicals in the ammoniumsubstituent correspond to the same definition as alkyl in R¹ and can bethe same or different.

The biphenyl radical of the definition of R² is advantageouslyp-diphenylyl.

The naphthyl radical in the definition of R² is advantageously2-naphthyl.

By a carboxyl-substituted hydroxyphenyl radical in the definition of R³,there is particularly to be understood 3-carboxy-4-hydroxyphenyl.

The dimethylthiazolyl radicals in the definition of R³ are connectedwith their 2-position to the tetrazolium salt. A preferreddimethylthiazolyl radical carries the methyl moieties in the 4- and5-positions.

Amongst the conventional counterions in the definition of A.sup.⊖ areespecially to be understood monovalent anions. For this purpose, thereare usually employed inorganic anions, for example halogen ions andpreferably chloride and bromide ions. In the case ofcarboxyl-substituted radicals R¹ or R³, The counteranion A.sup.⊖ canalso be the carboxylate ion.

The compounds listed in the following Table are especially preferred:

                  TABLE                                                           ______________________________________                                        tetra-                                                                        zolium                                                                        salt   R.sup.1     R.sup.2     R.sup.3                                        ______________________________________                                        T1     carboxyl    phenyl      2-carboxyphenyl                                T2     phenyl      phenyl      2-carboxyphenyl                                T3     2,4-dinitro-                                                                              phenyl      phenyl                                                phenyl                                                                 T4     carboxyl    phenyl      phenyl                                         T5      -p-(trimethyl-                                                                           phenyl      phenyl                                                ammonium)-                                                                    phenyl                                                                 T6     4-carboxy-  phenyl      phenyl                                                phenyl                                                                 T7      -p-nitrophenyl                                                                            -p-nitrophenyl                                                                           phenyl                                         T8     phenyl      phenyl      4,5-dimethyl-                                                                 thiazolyl                                      T9     hydrogen    phenyl      phenyl                                          T10    -n-butyl   phenyl      phenyl                                          T11   phenyl      2-naphthyl  phenyl                                          T12   phenyl       -p-diphenylyl                                                                            phenyl                                          T13   methyl      phenyl      3-carboxy-4-                                                                  hydroxyphenyl                                   T14   methyl      phenyl      phenyl                                          T15   ethyl       phenyl      phenyl                                          T16   4-carboxy-  phenyl      2-carboxyphenyl                                       phenyl                                                                  T17   2-carboxy-  phenyl      2-carboxyphenyl                                       phenyl                                                                 ______________________________________                                    

Compounds T1, T2, T3, T4, T5, T6 and T8 are quite especially preferred.

The concentration of the tetrazolium salts used for the removal ofdisturbances of the measurement reaction depends upon the amount ofreducing substances to be expected in the sample. As a rule, 0.001 to100 mmole/liter, preferably 0.01 to 20 mmole/liter and most preferably0.05 to 5 mmole/liter of tetrazolium salt is used.

The process according to the present invention for the determination ofsubstrates or of enzyme activities with the use of a redox reaction asmeasurement reaction and the addition of one or more tetrazolium saltsfor the removal of disturbing reducing substances can be carried out ata pH value of from 3 to 11, depending upon the enzymes required for theprocess in question. The pH range of from 6.5 to 9 is preferred. Asbuffer, there can be used any substance which has a sufficient buffercapacity in the necessary pH range. However, it is preferred to usephosphate or tris buffer. The concentration of the buffer can be from0.01 to 1 mole/liter and preferably from 0.05 to 0.2 mole/liter.

For a better solubility of the formazane formed from the tetrazoliumsalt added for removal of the disturbance and for increasing thereaction velocity of the reaction removing the disturbance, to thesample to be measured can optionally be added a non-ionic detergent, forexample Triton X-100, Tween 80 or polyvinylpyrrolidone. Theconcentration of such optionally added detergents depends upon thecritical micelle concentration of the detergent in question. As a rule,it is from 0.01 to 5% and especially from 0.05 to 0.5%.

In a preferred embodiment of the process according to the presentinvention, the disturbance-removing action of the tetrazolium salt canbe accelerated by the addition of one or more electron transmitters. Forthe acceleration of the disturbance-removing reaction and as adjuvant inthe oxidation of disturbing substances, there can be used a series ofelectron transmitters, examples of which includephenazine-methosulphate, phenazine-ethosulphate,8-dimethylamino-2,3-benzophenoxazine,1-methoxy-5-methylphenazinium-methyl sulphate and diaphorase. Of these,phenazine-ethosulphate and diaphorase are especially preferred.

In the case of the determination of substrates or of enzyme activitieswith the use of a redox reaction as measurement reaction, a certaindisturbance-removing effect can already be ascertained with electrontransmitters alone, without additional tetrazolium salt. However, theaction of the above-mentioned electron transmitters does not suffice fora substantial or even complete disturbance removal from the redoxmeasurement reaction. For this purpose, the combination according to thepresent invention with a tetrazolium salt is necessary.

The concentrations of the electron transmitters to be added to thesample to be freed from disturbance are preferably from 0.005 to 100mmole/liter and especially from 0.05 to 5 mmole/liter. In the case ofdiaphorase, there are preferably used amounts of from 0.001 to 100U/ml., the range of from 0.1 to 20 U/ml. being especially preferred.

The present invention also provides a reagent for the photometricdetermination of substrates or enzyme activities which contain a systemfor the determination of a substrate or of an enzyme with a redoxreaction as measurement reaction and, in addition, one or moretetrazolium salts which irreversibly, easily and quickly remove thedisturbing reducing substances under the conditions prevailing in thesubstrate or enzyme test in question and thereby make possible adisturbance-free measurement.

In principle, for such as reagent, all tetrazolium salts can be usedwhich do not absorb or only absorb to a negligible extent in thewavelength range in which the measurement reaction is to be monitored.In the case of many substrates and enzymes which are important inclinical chemistry, this wavelength range is from 500 to 600 nm.Therefore, in particular, tetrazolium salts of general formula (I) haveproved to be useful. Especially preferred in combination with the otherreagent components necessary for the determination are compounds T1 toT17. Of these, tetrazolium salts T1, T2, T3, T4, T5, T6 and T8 haveproved to be quite especially advantageous.

The reagent according to the present invention contains one or moretetrazolium salts, the concentration of which depends upon the amount ofdisturbing reducing substance to be expected. As a rule, it contains0.001 to 100 mmole/liter, preferably from 0.01 to 20 mmole/liter andespecially preferably from 0.05 to 5 mmole/liter.

In order, in the case of the determination of substrates or enzymeactivities with the use of a redox reaction as measurement reaction andwith the addition of one or more tetrazolium salts for the removal ofdisturbing reducing substances, to maintain a particular pH value forthe enzymes necessary, the reagent according to the present inventioncan contain a buffer. The pH of the reagent according to the presentinvention is in the range of from 3 to 11 and preferably in the range offrom 6.5 to 9. Buffers which can adjust such a value are, in principle,all those which lie with their pK value within the given pH range. Thereagent according to the present invention can contain any of thesebuffers. As preferred buffer, the reagent according to the presentinvention contains phosphate or tris buffer. The concentration of thebuffer in the reagent can be from 0.01 to 1 mole/liter and preferablyfrom 0.05 to 0.2 mole/liter.

For the better solubility of the formazane formed from the tetrazoliumsalt added for the removal of the disturbances and for increasing thereaction velocity of the disturbance-removing reaction, the reagentaccording to the present invention can optionally also contain anon-ionic detergent, for example Triton X-100, Tween 80 orpolyvinylpyrrolidone. The concentration of such detergents depends uponthe critical micelle concentration of the detergent in question. As arule, it is from 0.01 to 5% and especially from 0.05 to 0.5%.

As especially preferred reagent according to the present invention ischaracterised in that, apart from one or more tetrazolium salts, it alsocontains one or more electron transmitters. As such, there can be used,for example, phenazine-methosulphate, phenazine-ethosulphate,8-dimethylamino-2,3-benzophenoxazine,1-methoxy-5-methylphenazinium-methyl sulphate or diaphorase. Asespecially preferred electron transmitters, there have proved to beuseful phenazine-ethosulphate and/or diaphorase in combination with theother reagent components necessary for the determination of particularsubstrates or enzyme activities.

The concentration of the electron transmitters in the reagent is from0.005 to 100 mmole/liter, preferably from 0.05 to 5 mmole/liter. In thecase of diaphorase, the reagent preferably contains 0.001 to 100 U/ml.of the enzyme, the concentration range of from 0.1 to 20 U/ml. beingespecially preferred.

As system for the determination of a substrate or enzyme with a redoxreaction as measurement reaction, the reagent according to the presentinvention can contain, for example, for the determination of glucose,glucose oxidase, peroxidase, 4-aminophenazone and phenol; for thedetermination of uric acid, uricase, peroxidase,2-hydrazono-2-,3-dihydro-3-methyl-6-sulphobenzothiazole andN-ethyl-N-β-sulphoethyl-m-toluidine; and for the determination ofcholesterol, cholesterol esterase, cholesterol oxidase, peroxidase,4-aminophenazone and phenol.

The foregoing components as well as others, can be used in the inventionas described herein, to determine substrate or enzyme activity.

The reagent according to the present invention has proved to beespecially effective when, for the removal of disturbing reducingsubstances, it contains one or more tetrazolium salts and, as componentsof the system for the determination of a substrate or enzyme, itcontains a hydrogen peroxide-forming enzyme, for example theabove-mentioned glucose oxidase, uricase or cholesterol oxidase. In thesame way, the reagent according to the present invention has proved tobe especially advantageous when, for the measurement reaction, itcontains a phenol, for example phenol itself or2,4,6-tribromohydroxybenzoic acid, or an aniline derivative, for exampleN-ethyl-N-β-sulphoethyl-m-toluidine, which can be oxidatively coupledwith reagents such as 4-aminophenazone, 4-aminoantipyrine or2-hydrazono-2,3-dihydro-3-methyl-6-sulphobenzothiazole. Examples of suchredox reactions as measurement reactions for the determination ofglucose, uric acid and cholesterol have already been mentioned.

The present invention can also be used in the field of rapiddiagnostics. As a rule, such rapid diagnostics are contained in a kitwhich comprises the various reagents required for carrying out theprocess either impregnated into an absorbent, insoluble carrier, such aspaper, fleece or the like, or applied with an appropriate binding agentto a carrier film or a swellable film as coating.

In one embodiment, the reagent according to the present invention, whichcontains one or more tetrazolium salts for the removal of disturbingreducing substances, is impregnated on to an absorbent carrier, forexample paper. In this way, there are obtained test papers for thedetection of, for example, glucose, uric acid or cholesterol, which arepractically undisturbed by, for example, ascorbic acid.

However, according to the present invention, tetrazolium salts can alsobe applied to a separate carrier, combined with the carrier for theother reagents, for example laid thereover, stuck thereon and sealed intherewith. In such an embodiment, for example, a water-soluble paper(for example according to U.S. Pat. No. 4,061,468) can be impregnatedwith tetrazolium salt, whereas the system necessary for thedetermination of a substrate or of an enzyme activity is presentimpregnated on a water-insoluble, absorbent carrier material. Bothcarrier materials are advantageously so laid upon one another that thesample to be determined first contacts the tetrazolium salt and thencomes into contact with the water-insoluble carrier material.

In a preferred embodiment, separate zones of the carrier material canalso be impregnated with tetrazolium salt(s) and test reagents. In thiscase, the carrier is preferably brought into contact with the solutionto be investigated in such a manner that the solution first comes intocontact with the tetrazolium salt-containing zone and is sucked fromthere into the zone which contains the other necessary test reagents.

For the purpose of illustration of such an embodiment, in FIG. 1 of theaccompanying drawings there is shown an example of a device incross-section which, according to U.S. Pat. No. 4,477,575 permits, onthe one hand, the separation from whole blood of the serum or plasmanecessary for the test and, on the other hand, on the basis of a specialconstruction of the reagent and adjuvant material layers, permits atempering, pre-reaction and objective starting of the main reaction.

The device according to FIG. 1 is constructed as follows: on to an inertcarrier film 8 is fixed a transport fleece 7 consisting of glass fibres.This transport fleece 7 is partly covered by a fleece 5, also consistingof glass fibres, which is fixed by means of a fixing mesh 6. Between thefleece 5 and the transport fleece 7 is arranged a disturbance-freeingfabric 4 which is impregnated with materials which are able to removesubstances which disturb the measurement reaction. Laterally of thetransport fleece 7, via an adhesion point 9, there is fixed atransparent film 1 consisting of a transparent synthetic resin. Underthis transparent film 1 is arranged a reagent layer 2 which consists ofa swellable or absorbent film into which are incorporated the materialsnecessary for the measurement reaction. Under the reagent layer 2 isprovided a covering layer 3 which usually consists of a synthetic resinor gelatine film permeated with strongly reflecting substances, such asbarium sulphate, titanium dioxide or the like, and which serves as anoptically white background. Light shone in for observation purposes isthus completely remitted and possible discolorations of the fleece 7cannot be seen. Reagent layer 2 and covering layer 3 are togetherreferred to as the test layer.

If whole blood is applied to the fixing mesh 6, then this is separatedin the glass fibre fleece 5 into serum and erythrocytes, the latterbeing retained. Upon passing through the disturbance-freeing fabric 4,the reducing component materials disturbing the measurement reaction areremoved so that only serum passes over into the lefthand region of thetransport fleece 7, which serum does not contain any substancesdisturbing the measurement. After applying pressure to the transparentfilm 1, the measurement reaction is started after the serum hascompletely filled the transparent fleece 1. As a result of the pressurecontact, the serum penetrates through the covering layer 3 into thereagent layer 2 and moistens this uniformly. The reaction is observedthrough the transparent film on the basis of the coloration of thereagent layer 2.

Tetrazolium salts which can be used for the process and reagentaccording to the present invention include5-carboxy-3-(2-carboxyphenyl)-2-phenyl-2H-tetrazolium hydroxide internalsalt (T1), 3-(2-carboxyphenyl)-2,5-diphenyl-2H-tetrazolium hydroxideinternal salt (T2), 2,3-diphenyl-5-(2,4-dinitrophenyl)-2H-tetrazoliumchloride (T3), 2,3-diphenyl-5-[4-(trimethylammoniophenyl)-2H-tetrazoliumdichloride (T5), as well as3-(2-carboxyphenyl)-5-(4-carboxyphenyl)-2-phenyl-2H-tetrazoliumhydroxide internal salt (T16) and3,5-bis-(2-carboxyphenyl)-2-phenyl-2H-tetrazolium chloride (T17), whichare new compounds and are also the subject of the present invention.They can be represented by the general formula: ##STR3## wherein R^(1')is a carboxyl group or a phenyl radical which is unsubstituted orsubstituted in the 2- or 4-position by a carboxyl group, disubstitutedin the 2- and 4-positions by nitro groups or substituted in the4-position by a trimethylammonium radical, R^(2') is an unsubstitutedphenyl radical, R^(3') is a phenyl radical which is unsubstituted orsubstituted in the 2-position by a carboxyl group and A'.sup.⊖ is aconventional counterion.

The conventional counterions in the definition of A'.sup.⊖ correspond tothose given for A.sup.⊖ in general formula (I).

Apart from a good water-solubility, the compounds show, as required, nolight absorption in the wavelength range of from 500 to 600 nm and donot disturb the measurement reaction.

The preparation thereof takes place according to known methods which aredescribed, for example, by R. Kuhn and D. Jerchel, Ber. Dtsch. Chem.Ges., 74, 94/1941; D. Jerchel and W. Mohle, Ber. Dtsch. Chem. Ges., 77,600/1944 and R/ Wizinger and V. Bisro, Helv. Chim. Acta, 32, 909/1949.

In this case, aldehyde phenylhydrazones known from the literature of thegeneral formula: ##STR4## wherein R is an esterified carboxyl group, anunsubstituted phenyl radical or a phenyl radical substituted in the 2-or 4-position by a carboxyl group, in the 2- and 4-positions by nitrogroups or in the 4-position by a trimethylammonium group and R^(2') isan unsubstituted phenyl radical, are converted with diazonium salts ofthe general formula: ##STR5## wherein R^(3') is an unsubstituted phenylradical or a phenyl radical substituted in the 2-position by a carboxylgroup and A'.sup.⊖ is a conventional counterion, by alkaline couplinginto formazanes of the general formula: ##STR6## wherein R and R^(2')have the meanings given in general formula (II) and R^(3') has themeaning given in general formula (III).

An esterified carboxyl group in the definition of R is hereby preferablya lower alkyl esterified carboxyl group, lower alkyl hereby meaning a C₁-C₄ -hydrocarbon radical, the methyl and ethyl esters being especiallypreferred.

Conventional counterions of the definition of A'.sup.⊖ are the same arethose given for A.sup.⊖ in general formula (I).

As basic reaction medium for the alkaline azo coupling, there can beused, for example, pyridine, sodium acetate dissolved indimethylformamide and/or an alcohol or an alcoholic solution of analkali metal hydroxide. Alcohols are hereby to be understood to be loweraliphatic alcohols, methanol and ethanol being preferred.

The conversion of formazanes of general formula (IV) into tetrazoliumsalts of general formula (I') takes place by oxidation either by meansof isopentyl nitrite in alcoholic hydrochloric acid analogously to themethod described by D. Jerchel and H. Fischer, Liebigs Ann. Chem., 563,200/1949, or by means of lead tetraacetate analogously to the methoddescribed by R. Kuhn and D. Jerchel, Ber. Dtsch. Chem. Ges., 74,941/1941 or by D. Jerchel and H. Fischer, Liebigs Ann. Chem., 563,200/1949 in chloroform.

The amyl nitrite method hereby has the advantage that a laborious columnchromatographic post-purification can be omitted. However, because ofthe low oxidation potential of the nitrite, it is not of universalapplicability.

Since the double bonds and the positive charge of the tetrazolium ringcannot be localised within the nitrogen system, for the preparation ofthe compounds according to the present invention of general formula (I')there can, of course, also be used as starting materials analogouslyformazanes of the general formula: ##STR7## wherein R, R^(2') and R^(3')have the same meanings as in the definitions given in general formula(IV).

Formazanes of general formula (IV') can be prepared analogously to thedescribed process from aldehyde phenylhydrazones of the general formula:##STR8## by reaction with diazonium salts of the general formula:##STR9## The symbols used in general formulae (II') and (III') have thesame meanings as in general formulae (II) and (III).

The following Examples are given for the purpose of further illustratingthe present invention:

EXAMPLE 1 Preparation of Tetrazolium Salts T1, T2, T3, T5, T16 and T17a) Preparation of the starting formazanes

0.1 mole of amine is suspended in 100 ml. water and, after the additionof 0.3 mole 12N hydrochloric acid, diazotised by the dropwise additionof a concentrated aqueous solution of 1.04 mole sodium nitrite at 0° to5° C. Within the course of 30 minutes, the cooled diazonium saltsolution is added dropwise, with stirring and cooling to below 10° C.,to a solution of 0.1 mole aldehyde phenylhydrazone and 0.35 mole sodiumacetate (or 100 ml. pyridine) in 180 ml. ethanol and 100 ml.Dimethylformamid and subsequently stirred for 1 hour. The crystallineslurry formed is filtered off with suction, well washed with water andthereafter with a little methanol and then dried. A purification of theformazane thus obtained can be carried out by recrystallization fromglacial acetic acid, methanol-water or from dimethylformamide-water orby column chromatography on silica gel 60 (Merck) with methylenechloride-methanol (5:1 v/v) or methylene chloride as eluent. Thefollowing formazanes are obtained according to this method:

    ______________________________________                                                                      azo                                                   starting                coup-                                                 com-                    ling                                            form- pound    yield  m.p.    in     purification                             azane for      (%)    (°C.)                                                                          medium method                                   ______________________________________                                        F1    T1       40     180     A      recrystallization                                              (decomp.)      from methanol/                                                                water                                    F2    T2       40     187     C      Wizinger and                                                   (decomp.)      Bisro, Helv.                                                                  Chim. Acta, 32,                                                               910/1949                                 F3    T3       62     204     B      column chromato-                                               (decomp.)      graphy on silica                                                              gel, eluent:                                                                  methylene                                                                     chloride                                 F5    T5       70     187     A      stirring with                                                  (decomp.)      isopropanol                               F16   T16     35     >300    B      column chromato-                                               (decomp.)      graphy in silica                                                              gel, eluent:                                                                  methylene                                                                     chloride/                                                                     methanol                                                                      (5:1 v/v)                                 F17   T17     50     191     A      column chromato-                                               (decomp.)      graphy on silica                                                              gel, eluent:                                                                  methylene                                                                     chloride/                                                                     methanol                                                                      (5:1 v/v)                                ______________________________________                                         medium A = dimethylformamide/sodium acetate                                   medium B = dimethylformamide/pyridine                                         medium C = ethanolic lye (sodium or potassium hydroxide)                      F1 = 2(3-carboxyethyl-5-phenyl-1-formazano)-benzoic acid                      F2 = 2(3,5-diphenyl-1-formazano)-benzoic acid                                 F3 = 1,5diphenyl-3-(2,4-dinitrophenyl)-formazane                              F5 = 4(1,5-diphenyl-3-formazano)-N,N,N-trimethylammonium chloride             F16 = 2,4(5-phenyl-1,3-formazandilyl)-bis-benzoic acid                        F17 = 2,2(5-phenyl-1,3-formazandilyl)-bis-benzoic acid.                  

b) Oxidation of the formazanes A) Lead tetraacetate method

0.1 mole of formazane is dissolved in anhydrous chloroform and mixedwith 0.12 mole lead tetraacetate. The reaction mixture is stirred for 30minutes and then insoluble material is filtered off with suction. Thefiltrate is evaporated, the residue is mixed with water and thenfiltered off with suction and the filtrate is acidified with 2Nhydrochloric acid. The lead chloride thus precipitated is filtered offwith suction, the filtrate is evaporated and the residue isrecrystallised from ethanol or purified by column chromatography onsilica gel 60 (Merck) with the use of chloroform-methanol (19:1 v/v),chloroform-methylene chloride (5:1 v/v) or methylene chloride-methanol(5:1 v/v) as elution agent.

B) Isopentyl nitrite method

0.1 mole of formazane is suspended into the 10 to 20 fold amount ofethanol, 0.2 mole isopentyl nitrite is added thereto and 0.15 moleethanolic hydrochloric acid is added dropwise thereto within the courseof 20 minutes, while stirring. Subsequently, the reaction mixture isfurther stirred for 1 hour. If the reaction mixture is not yetdecolorised, for complete decolorisation it is heated to 40° C. Thetetrazolium salt is precipitated out by the addition of diethyl etherand then filtered off with suction and dried. For purification, theproduct can be stirred up with methanol or isopropanol, filtered offwith suction and dried. Recrystallization can be carried out frommethanol, methanol-water or glacial acetic acid. For the purification ofthe tetrazolium salt, it is advantageous to use column chromatography onsilica gel 60 (Merck) with chloroform-methanol (19:1 v/v),chloroform-methylene chloride (5:1 v/v) or methylene chloride-methanol(5:1 v/v).

The following tetrazolium salts are prepared according to one of themethods A) or B):

    ______________________________________                                                               method                                                 tetra                  of                                                     zolium                                                                              yield  m.p.      oxida-                                                                              method of                                        salt  (%)    (°C.)                                                                            tion  purification                                     ______________________________________                                        T1    35     184       A     column chromatography on                                      (decomp.)       silica gel; eluent:                                                           chloroform/methanol                              T2    48     259       A     recrystallization from                                        (decomp.)       ethanol/water                                    T3    47     246       A     column chromatography on                                      (decomp.)       silica gel; eluent:                                                           methylene chloride-                                                           methanol (5:1 v/v)                               T5    82     162       A     stirring up with methanol                                     (decomp.)                                                         T16  42     >300      A     column chromatography on                                      (decomp.)       silica gel; eluent:                                                           methylene chloride-                                                           methanol (5:1 v/v)                                T17  74     230       B     column chromatography on                                      (decomp.)       silica gel; eluent                                                            methylene chloride-                                                           methanol (5:1 v/v)                               ______________________________________                                         oxidation method A: lead tetraacetatechloroform                               oxidation method B: isopentyl nitriteethanolic hydrochloric acid              T1 = 5carboxy-3-(2-carboxyphenyl)-2-phenyl-2H-tetrazolium hydroxide,          internal salt                                                                 T2 = 3(2-carboxyphenyl)-2,5-diphenyl-2H-tetrazolium hydroxide, internal       salt                                                                          T3 = 2,3diphenyl-5-(2,3-dinitrophenyl)-2H-tetrazolium chloride                T5 =  2,3diphenyl-5-[4(timethylaminophenyl)-2H-tetrazolium dichloride         T16 = 3(2-carboxyphenyl)-5-(4-carboxyphenyl)-2-phenyl-2H-tetrazolium          hydroxide, internal salt                                                      T17 = 3,5bis-(2-carboxyphenyl)-2-phenyl-2H-tetrazolium chloride.         

EXAMPLE 2 Uric acid determination in the presence of various disturbingsubstances

Into cuvettes A, B and C with a layer thickness of 1 cm. are pipettedthe following solutions:

    ______________________________________                                                     A        B       C                                               ______________________________________                                        sample          50 μl.  50 μl.                                                                             50 μl.                                  disturbing substance*                                                                        -          +       +                                           tetrazolium salt*                                                                            -          -       175 μl.                                  and/or electron                                                               transmitter                                                                   0.1M potassium phosphate                                                                     175 μl. 175 μl.                                                                            -                                           (pH 8)                                                                        ______________________________________                                         *Type and concentration are given in the following Examples 2a)-2h).     

The solutions pipetted into the cuvettes are mixed and incubated for 1minute at 25° C. There then follows the addition of 2 ml. uric acidreagent of the following composition:

potassium phosphate, 0.1 mole/liter, pH 8

2,4,6-tribromohydroxybenzoic acid, 20 mmole/liter

4-aminoantipyrine, 0.1 mmole/liter

sodium azide, 1 g/liter

peroxidase, 4U/ml.

uricase, 2 U/ml.

After incubation for 5 minutes at 25° C., the extinctions of each of thereaction mixtures is measured at a wavelength of 546 nm against areagent blank (without sample). For calibration, instead of a sample tobe measured, there is used an aqueous uric acid standard (6 mg./dl.).

The uric acid concentrated measured in cuvette A (without disturbingsubstance) is taken as being 100%. The uric acid finding rate in thepresence of disturbing substances is determined experimentally in thefollowing Example 2a) to 2h). The results demonstrate that the findingrate in cuvette series C (with tetrazolium salt) is distinctly higherthan without the addition (cuvette series B).

The concentrations of the tetrazolium salts and/or electron transmittersgiven in the following refer to the end concentration in the solution tobe measured after the addition of these substances and of buffer. Theconcentration of the disturbing substance refers to the concentration inthe sample before the addition of buffer, tetrazolium salt and/orelectron transmitter.

a) Disturbing substance: ascorbate 3 mg./dl.

T3, 0.4 mmole/liter

phenazine-ethosulphate (PES): 20 μmole/liter

    ______________________________________                                                           % finding in                                                                  B    C                                                     ______________________________________                                        human serum 1        61     100                                               human serum 2        65     100                                               human serum 3        62      92                                               human serum 4        58     110                                               human serum 5        83     105                                               control serum PPU Ch. 1                                                                            80     101                                               control serum PPU Ch. 2                                                                            74     104                                               ______________________________________                                    

b) Disturbing substance: bilirubin

T8: 0.4 mmole/liter

PES: 20 μmole/liter

    ______________________________________                                        bilirubin concentration                                                       in the sample (human                                                                             % finding in                                               serum)             B      C                                                   ______________________________________                                        100 mg./liter      76     89                                                  200 mg./liter      63     86                                                  ______________________________________                                    

c) Disturbing substance: α-methyl-DOPA 20 mg./liter

T3 or T5: 0.6 mmole/liter

PES: 25 μmol/liter

sample: uric acid standard 6 mg./dl.

    ______________________________________                                                        % finding in                                                  tetrazolium salt  B      C                                                    ______________________________________                                        T3                62     94                                                   T5                62     94                                                   ______________________________________                                    

d) Disturbing substance: dobesilate calcium 25 mg./liter

T3: 0.6 mmole/liter

PES: 25 μmole/liter

    ______________________________________                                                % finding in                                                                  B    C                                                                ______________________________________                                                90   98                                                               ______________________________________                                    

e) Disturbing substance: ascorbate 3 mg./dl

tetrazolium salt: 0.06 mmole/liter

PES: 25 μmole/liter

sample: uric acid standard 6 mg./dl.

    ______________________________________                                                        % finding in                                                  tetrazolium salt  B      C                                                    ______________________________________                                        T1                39     82                                                   T2                39     88                                                   T3                39     82                                                   T4                39     89                                                   T5                39     94                                                   T6                39     93                                                   ______________________________________                                    

f) Disturbing substance: ascorbate 3 mg./dl.,

T8: 0.4 mmole/liter

PES: 20 μmole/liter

sample: human serum

    ______________________________________                                                % finding in                                                                  B    C                                                                ______________________________________                                                72   93                                                               ______________________________________                                    

g) Disturbing substance: ascorbate 3 mg./dl.

T8: 0.4 mg./liter

diaphorase: 0.4 U/ml.

sample: human serum

    ______________________________________                                                % finding in                                                                  B    C                                                                ______________________________________                                                65   96                                                               ______________________________________                                    

h) Disturbing substance: ascorbate 17.6 mg./dl.

PES: 25 μmole/liter

sample: uric acid standard 6 mg./dl.

    ______________________________________                                                % finding in                                                                  B    C                                                                ______________________________________                                                6    53                                                               ______________________________________                                    

EXAMPLE 3 Glucose Determination in the Presence of Ascorbic Acid

Into 1 cm path length cuvettes A, B and C are pipetted the followingsolutions:

    ______________________________________                                                     A        B       C                                               ______________________________________                                        sample          50 μl.  50 μl.                                                                             50 μl.                                  ascorbic acid  -          +       +                                           tetrazolium salt (T3)                                                                        -          -       175 μl.                                  and electron transmitter                                                      (phenazine-ethosulphate                                                       (PES))                                                                        0.1M potassium phosphate                                                                     175 μl. 175 μl.                                                                            -                                           buffer (pH 7.8)                                                               ______________________________________                                    

The solutions pipetted into the cuvettes are mixed and incubated for 5minutes at 25° C. There is then added 2 ml. of glucose reagent of thefollowing composition:

    ______________________________________                                        tris-phosphate buffer (pH 7.8)                                                                    180     mmole/liter                                       phenol              11      mmole/liter                                       3,4-dichlorophenol  2.1     mmole/liter                                       fatty alcohol polyglycol ether                                                                    0.24%                                                     4-aminophenazone    0.8     mmole/liter                                       peroxidase          1       U/ml.                                             glucose oxidase     15      U/ml.                                             ______________________________________                                    

After incubation at 25° C. for 30 minutes, the extinctions of thereaction mixtures are measured at a wavelength of 546 nm against areaction blank (without sample). For calibration, instead of a sample tobe measured there is used an aqueous glucose standard (100 mg./dl.).

The glucose concentration measured in cuvette A (without disturbingsubstance) is taken as being 100%. The glucose finding rate in thepresence of disturbing substances is determined experimentally in thefollowing Examples 3a) and 3b). The results demonstrate that the findingrate in cuvette series C (with tetrazolium salt) is clearly higher thanwithout addition (cuvette series B).

The concentration of the tetrazolium salt T3 and of the electrontransmitter PES given in the following concern the end concentrations inthe solution to be measured after the addition of these substances andof the buffer. The concentration of the disturbing substance ascorbicacid is, in each case, the concentration in the sample before theaddition of buffer, T3 and PES.

a) glucose concentration: 25 mg./dl.

disturbing substance: ascorbate

T3: 0.25 mmole/liter

PES: 20 μmole/liter

    ______________________________________                                                          % finding in                                                ascorbate concentration                                                                           B      C                                                  ______________________________________                                         3 mg./dl.          65     94                                                  6 mg./dl.          27     91                                                 12 mg./dl.           0     85                                                 ______________________________________                                    

b) glucose concentration: 50 mg./dl.

disturbing substance: ascorbate 3 mg./dl.

T3: 0.25 mmole/liter

PES: 20 μmole/liter

    ______________________________________                                                % finding in                                                                  B    C                                                                ______________________________________                                                83   94                                                               ______________________________________                                    

EXAMPLE 4 Test System for the Determination of Uric Acid in Blood a)Test Layer for the Detection of Uric Acid

From the components set out below, there is produced a coating masswhich is raked out on to a transparent film 1 in a wet film thickness of200 μm and then dried:

18 g. of a synthetic resin dispersion of a mixed polymer of vinylacetate and vinyl propionate; 1.38 g. of alginate; 69 g. of a 0.45Mtris-citrate buffer (pH 7.5); 0.47 g. of indicator(2-(3,5-dimethoxy-4-hydroxyphenyl)-4-(5)-(4-dimethylaminophenyl)-5-(4)-methyl-(1H)-imidazolehydrochloride); 0.025 g. 1-(3-chlorophenyl)semicarbazide; 0.025 g.monomagnesium dipotassium ethylenediamine-tetraacetate dihydrate; 0.5 g.Triton X100; 0.6 g. hexanol; 200 KU peroxidase and 2 KU uricase.

On to the so produced reagent layer 2 there is placed a covering layer 3as an optical white background of the following composition which israked on with a layer thickness of 200 μm and dried; 52 ml. 0.1Mtris-citrate buffer (pH 7.0); 5.5 g. titanium dioxide; 2.7 g.diatomaceous earth; 0.4 g. alginate; 1.4 g. of a synthetic resindispersion of a mixed polymer of vinyl acetate and vinyl propionate and0.2 g. Triton X100.

b) Disturbance-removal Fabric 4

Nylon fabric is impregnated with a solution of the following compositionand dried at 60° C.: 5 mg. 5-(2,4-dinitrophenyl)-2,3-diphenyltetrazoliumchloride (T3); 5 mg. phenazine-ethosulphate; 30 mg.dioctylsulphosuccinate sodium; ad 100 ml. 0.5M phosphate buffer (pH 7.5)

As material for a comparative test, the same fabric was impregnated withan appropriate solution which only contained buffer and wetting agentbut no tetrazolium salt or phenazine-ethosulphate.

c) Total System

The above-described components are used to produce a test systemaccording to FIG. 1 of the accompanying drawings. The test can be usedfor the determination of uric acid in blood, plasma and serum.

d) Carrying Out of the Test

For the determination of uric acid, 30 μl. of serum are applied to thefixing mesh 6, after 1 minute the transparent film 1 is pressed downand, after a further 2 minutes, the color formed is measured with aremission photometer and the uric acid values determined from apreviously produced calibration curve.

A serum sample with a uric acid content of 6.2 mg./dl. is divided intotwo. Ascorbic acid is added to one part so that a content of 2 mg./dl.results. Both serum samples are investigated not only with the testsystem containing the tetrazolium salt but also with the test systemwithout tetrazolium salt, which serves as a comparison.

The following uric acid values were measured in mg./dl.:

    ______________________________________                                                      serum without                                                                           serum with                                                          ascorbic acid                                                                           ascorbic acid                                         ______________________________________                                        with tetrazolium salt                                                                         6.27        6.09                                              without tetrazolium salt                                                                      6.02        4.61                                              ______________________________________                                    

List of References

1=transparent film

2=reagent layer

3=covering layer

4=disturbance-removal fabric

5=glass fibre fleece

6=fixing mesh

7=transport fleece

8=carrier film

9=point of adhesion

While there have been described what are at present considered to be thepreferred embodiments of this invention, it will be obvious to oneskilled in the art that various changes and modifications may be madetherein without departing from the invention, and it is, therefore,aimed to cover all such changes and modifications as fall within thetrue spirit and scope of the invention.

We claim:
 1. Reagent kit for determining an enzyme substrate or enzymeactivity in a sample by measuring at a measurement wavelength lightabsorption of a photometrically measurable material formed by a redoxreaction of a redox color indicator system or light absorption of saidcolor indicator system after that reaction, said kit comprisinga redoxreagent, color indicator for the photometrical determination of thesubstrate or enzyme activity, and one or more tetrazolium compounds ofthe formula ##STR10## in which R¹ is a hydrogen atom, a carboxyl groupor an alkyl, phenyl, nitrophenyl, dinitrophenyl, carboxyl-substitutedphenyl or trialkylammoniumphenyl radical, R² is a phenyl, nitrophenylbiphenyl or naphthyl radical, R³ is a phenyl, carboxyl-substitutedphenyl, carboxyl-substituted hydroxyphenyl or dimethylthiazolyl radical,and A⁻ is a monovalent anion, which tetrazolium compound, by reactionwith reducing substances in the sample which would disturb saidmeasurement of said light absorption, forms a formazane which does notabsorb light at all or absorbs light only to a negligible extent thatdoes not interfere with said measuring of light absorption at themeasurement wavelength.
 2. Reagent kit according to claim 1,additionally comprising one or more electron transmitter, saidtetrazolium compounds and electron transmitters separated from eachother.
 3. Reagent kit of claim 1, wherein said tetrazolium compounds aresalts of 5-carboxy-3-(2-carboxyphenyl)-2-phenyl-2H-tetrazole,3-(2-carboxyphenyl)-2,5-diphenyl-2H-tetrazole,2,3-diphenyl-5-(2,4-dinitrophenyl)-2H-tetrazole,2,3-diphenyl-5-carboxy-2H-tetrazole,2,3-diphenyl)-5-[4-(trimethylammoniophenyl)]-2H-tetrazole,2,3-diphenyl-5-(4-carboxyphenyl)-2H-tetrazole or3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2-2-H-tetrazole.
 4. Reagent kitof claim 2, wherein said electron transmitters are selected from thegroup of phenazine-methosulphate, phenazine-ethosulphate,8-dimethylamino-2,3-benzophenoxazine,1-methoxy-5-methyl-phenazinium-methylsulphate or disphorase.
 5. A methodof removing disturbing reducing substances from an enzyme substrate orenzyme sample to be analyzed by a photometrically detectible redoxreaction of redox color indicator system, wherein light absorption of aphotometrically measurable material formed by a redox reaction or lightabsorption of said color indicator system after said reaction ismeasured at a measurement wavelength, which method comprises adding tosaid sample a tetrazolium salt which, by reaction with the reducingsubstances, forms a formazane which does not absorb light at all orabsorbs light only to a negligible extent that does not interfere withmeasuring the light absorption resulting from the redox reaction at themeasurement wavelength.
 6. A method according to claim 5 in which thetetrazolium salt is ##STR11## in which R¹ is a hydrogen atom, a carboxylgroup or an alkyl, phenyl, nitrophenyl, dinitrophenyl,carboxyl-substituted phenyl or trialkylammoniumphenyl radical,R² is aphenyl, nitrophenyl, biphenylyl or naphthyl radical, R³ is a phenyl,carboxyl-substituted phenyl, carboxyl-substituted hydroxyphenyl ordimethylthiazolyl radical, and A⁻ is a monovalent anion.
 7. A methodaccording to claim 6 in which the tetrazolium salt is a salt of5-carboxy-3-(2-carboxyphenyl)-2-phenyl-2H-tetrazole,3-(2-carboxyphenyl)-2,5-diphenyl-2H-tetrazole,2,3-diphenyl-5-(2,4-dinitrophenyl)-2H-tetrazole,2,3-diphenyl-5-carboxyl-2H-tetrazole,2,3-diphenyl-5-[4-(trimethylammoniophenyl)]-2H-tetrazole,2,3-diphenyl-5-(4-carboxyphenyl)-2H-tetrazole or3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazole.